Therapeutic potential of nanoceria pretreatment in preventing the development of urological chronic pelvic pain syndrome: Immunomodulation via reactive oxygen species scavenging and SerpinB2 downregulation

Abstract Urological chronic pelvic pain syndrome (UCPPS) manifests as pelvic pain with frequent urination and has a 10% prevalence rate without effective therapy. Nanoceria (cerium oxide nanoparticles [CNPs]) were synthesized in this study to achieve potential long‐term pain relief, using a commonly used UCPPS mouse model with cyclophosphamide‐induced cystitis. Transcriptome sequencing analysis revealed that serpin family B member 2 (SerpinB2) was the most upregulated marker in mouse bladder, and SerpinB2 was downregulated with CNP pretreatment. The transcriptome sequencing analysis results agreed with quantitative polymerase chain reaction and western blot analysis results for the expression of related mRNAs and proteins. Analysis of Gene Expression Omnibus (GEO) datasets revealed that SerpinB2 was a differentially upregulated gene in human UCPPS. In vitro SerpinB2 knockdown downregulated proinflammatory chemokine expression (chemokine receptor CXCR3 and C‐X‐C motif chemokine ligand 10) upon treatment with 4‐hydroperoxycyclophosphamide. In conclusion, CNP pretreatment may prevent the development of UCPPS, and reactive oxygen species (ROS) scavenging and SerpinB2 downregulation may modulate the immune response in UCPPS.


| INTRODUCTION
Urological chronic pelvic pain syndrome (UCPPS), a debilitating condition of chronic visceral pain, is characterized by chronic lower abdominal and pelvic pain, and frequent and urgent urination, which causes disability and has an unknown etiology. 1 Autoimmune processes, an inadequate glycosaminoglycan layer constitution, unspecified infection, toxic urinary constituents, and central neurogenic processes are some of the pathophysiological factors found in UCPPS. 1 Chronic inflammation and serial induction play integral roles in UCPPS progression. 2,3 UCPPS is prevalent in approximately 10% of the general US population. 4 UCPPS constitutes a significant socioeconomic impact of more than US$ 70 billion annually, while the treatment costs for UCPPS accelerate at a compound annual growth rate of approximately 5%. In addition to healthcare costs, UCPPS negatively influences psychosocial health, causing mental health issues such as sleep disturbance, anxiety, and depression. 5 The cyclophosphamide (CYP)-induced chronic cystitis mouse model, where mice are exposed to fourfold serial low-dose CYP induction, is the most commonly used mouse model to study UCPPS according to the Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network. 3  Administration-approved orally administered drug used for the treatment of UCPPS. Elmiron ® is believed to adhere to the bladder mucosal lining, thus maintaining or enhancing the urothelial permeability barrier. 8 However, Elmiron ® is a blood thinner and might increase the risk of bruising and bleeding. This treatment modality is listed as secondline treatment by the American Urological Association, requires longterm oral administration, and has low efficacy. 9 Hyaluronic acid (HA) has been recently used to treat UCPPS via intravesical instillation.
HA is a promising treatment, owing to its excellent biocompatibility and lack of biotoxicity. 10 However, urination accelerates the loss of HA; thus, patients require monthly installations, which increases the discomfort and costs associated with the treatment. 11 Cerium oxide (CeO 2 ) nanoparticles (nanoceria, CNPs) display several promising benefits for the treatment of UCPPS, such as low cost, fewer adverse effects, and distinctive mechanisms of action in the management of chronic pain. 12 CNPs demonstrate remarkable antioxidant properties by scavenging hydroxyl radicals, peroxynitrite, nitric oxide, and other free radicals and remaining active in tissues for prolonged periods via spontaneous redox switches. 13 CNPs help in preventing or treating numerous acute and chronic conditions, including inactivation of human coronavirus, 14 neuroprotection, 15 antidiabetic properties, 16 wound healing, 17 and ovarian cancer. 18 Hence, the requirement of CNPs for the prevention and treatment of chronic inflammation is rapidly increasing. Moreover, elucidating the role of serpin family B member 2 (SerpinB2), a protein implicated in the interaction between inflammation and cellular aging, is a priority. 19 Mouse SerpinB2 mRNA is detected mainly in the urinary bladder, skin, thymus, bone marrow, spleen, and lung. SerpinB2 has been associated with asthma 20 and chronic kidney disease. 21 However, its potential involvement in the development of UCPPS has not been described.
Therefore, in this study, we aimed to investigate the in vivo preventative and therapeutic effects of CNPs in CYP-induced chronic cystitis, and the molecular mechanism underlying SerpinB2 activity in UCPPS.

| Characterization of CNPs
The mean CNP diameter (22.53 nm, n = 10; n is the number of particles measured) was measured using scanning electron microscopy (SEM) and Nano Measurer software and the CNP particles were cube/octahedron-like in shape ( Figure 1a). The larger nanoparticles may be due to the aggregation of the smaller ones, as the exposed surface was unstable, because the SEM and transmission electron microscopy (TEM) images indicated that the grain size of CNP was around 20 nm 10 (Figure 1a In the present study, we synthesized CNPs using a slight modification of the metal-salt hydrolysis method. 22 While the most abundant element in the rare-earth family, cerium has two stable oxidation states, Ce 3+ and Ce 4+ (Figure 1e,f). The metal-salt hydrolysis method of synthesizing CNPs at room temperature 22 is more conducive to future laboratory production or the industrial mass production of this material. The coexistence of Ce 3+ and Ce 4+ results in self-regenerative antioxidant ability and long-term therapeutic potential. and Tnfα were significantly upregulated by 4-HC; however, they were downregulated in the CNP + 4-HC group. In the CNP groups, the cells did not exhibit excessive expression of inflammation-related genes, confirming that the application of 5 μg/ml CNP for 24 h does not induce cell inflammation. The values are presented as mean ± standard deviation. Data were compared via one-way analysis of variance with Tukey's post hoc tests. *p < 0.05, **p < 0.01, ***p < 0.001, p > 0.05: no significant difference (n.s.), n = 3. CNP, cerium oxide nanoparticles excessive ROS generated in the osteoarthritis joint in vivo and in vitro using the osteoarthritis-mimicking chondrocytes/macrophages co-culture models, where the immunomodulatory role of CNPs was shown to be the underlying molecular mechanisms. 26

| Behavioral and histological characterization of CYP-induced cystitis and the pretreatment effects of CNPs
Animal experimentation protocol is shown in Figure 3a. Animals with CYP-induced cystitis showed decreased rearing activity and pain threshold. In contrast, an opposite trend was observed in these parameters in the CNP pretreatment group (n = 3 per group, *p < 0.05), as shown in Figure 3b,c. Animals with CYP-induced cystitis exhibited an increased voiding frequency, which was alleviated in the CNP pretreatment group (n = 3 per group, *p < 0.05), as shown in and is a multicolony stimulating factor for granulocytes, macrophages, erythroid cells, and mast cells. 35 Serum levels of IL-6 and IL-3 were higher in the CYP cystitis group than in the control and CNP pretreatment groups (n = 3 in each group) using a Quantibody Multiplex ELISA array (RayBiotech) (Figure 4d). The results revealed that CNP successfully decreased pelvic pain and bladder inflammation among mice The clinical characteristics of UCPPS include familial clustering and elevated recurrence risk 36 ; therefore, CNP pretreatment may be considered to prevent the occurrence of familial clustering and the frequent recurrence of UCPPS. Currently, the concept of multimodal analgesia is broadly credited in the management of visceral pain. 37 Adverse events related to most affordable nonopioid analgesics (e.g., nonselective nonsteroid anti-inflammatory drugs, cyclooxygenase-2 inhibitors, alpha-2 agonists, ketamine, and local anesthetics) increase the risk of bleeding, peptic ulcer disease, and kidney injury. 38   The expression of UCPPS-related genes in the urinary bladder and urine of human subjects with UCPPS was substantially increased compared with that of subjects in the control group. The results revealed that 30 genes in the non-Hunner type cystitis group were collaborated between GSE11783 42 and GSE28242, 43 in which 17 genes were upregulated, including SerpinB2, and 13 genes were downregulated in the non-Hunner type cystitis group in contrast to the normal control group (Figure 5a,b). Enrichment analysis of GO terms of upregulated BP was shown in Figure S4 Non-Hunner type cystitis was considered a common finding, accounting for 90% of cases with poor overall treatment outcomes. 48 SerpinB2 was upregulated in non-Hunner type lesions in both humans and the CYP cystitis mouse model. SerpinB2, also known as plasminogen activator inhibitor 2 type A, expression was substantially downregulated in mice pretreated with CNP. SerpinB2 is the hub gene in human UCPPS pathogenesis based on protein-protein interactions ( Figure S5). SerpinB2 affects the ordinary fibrinolytic process in thrombus resolution. 49 Dysregulated SerpinB2 expression is associated with pre-eclampsia, lupus, asthma, scleroderma, periodontitis, 50

| Synthesis of cerium oxide nanoparticles
CNPs were synthesized using a slight modification of a previously described method. 22  (v/v) fetal bovine serum, and 1% (v/v) penicillin/streptomycin. The cells were maintained at 37 C in a humidified incubator containing 5% CO 2 . 52 In the present study, 4-HC was applied in vitro to induce T24 cells to produce oxidative stress and apoptosis. In an aqueous solution, 4-HC can rapidly hydrolyze to 4-hydroxycyclophosphamide, the same intermediate product resulting from the microsomal activation of CYP, 53 and therefore, 4-HC was used instead of CYP because of the need of CYP for microsomal activation. To assess the effects of CNP treatment in the UCPPS model established in vitro, T24 cells were subdivided into four groups: control, 4-HC, CNP pretreatment, and only CNP groups. The methods used in the water-soluble tetrazolium salt-1 and DCFDA cellular ROS detection assays are provided in the supplementary materials S1.

| Experimental animals: ICR mice with cyclophosphamide-induced cystitis
The study protocol was approved by the Institutional Animal Ethics Committee of our university (IACUC approval no. 110065) as per the National Research Council's Guide for the Care and Use of Laboratory Animals. The study was carried out using male ICR mice (age, 6-8 weeks; weight, 25-30 g). The animals were housed in a climatecontrolled environment at approximately 24 ± 2 C with a 12/12-h light/dark cycle and relative humidity of 55% ± 15% during the entire experimental period. They were observed for any signs of disease and maintained under standard conditions until the end of the experiment.

| Experimental group design
The experimental animals were subdivided into three groups: control,

| Mechanical pain threshold testing
The mechanical pain threshold was evaluated using Semmes-Weinstein monofilaments (Ugo Basile, Comerio, Italy). 54 The mice were assessed in isolated cages with a stainless-steel wire grid floor.

| Quantitative real-time PCR assay
The bladders of the mice were snap-frozen on CO 2 (n = 3 in each group). Total RNA was extracted from the bladders and cells using a TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase was used as an endogenous control. cDNA was synthesized using the SensiFAST cDNA Synthesis kit (Bioline Ltd., London, UK) with random hexamer and polymer (dT) primers. PCR assays were performed using a qPCR system (StepOne™ Software version 2.2; Thermo Fisher Scientific, Waltham, MA, USA). 56 Each sample was evaluated in triplicate for both the target gene and endogenous control. The mean cycle threshold (Ct) values of the triplicate samples were used for additional analyses. The Ct value for the target gene obtained from each sample was normalized to that of α-tubulin, an internal reference gene, and transformed to relative gene expression values using the 2 ÀΔΔCt method. The primer sequences are listed in Table S1, and the methods of RNA sequencing are provided in the Supplementary Methods S1. according to the manufacturers' instructions. 58 The sequences of SerpinB2 siRNA and the negative control are shown in Table S1.
T24 cells were seeded into a 96-well plate at a density of 10 4 cells/well and incubated for 24 h until full adhesion was achieved. The siRNA was initially transfected. Next, 37.5 μM of 4-HC was added to the medium and incubated for 4 h.

| Statistical analysis
Data were collected from three separate experiments and expressed as mean ± standard deviation or the mean ± square error of the mean.
The normality of distribution of the data was evaluated using the Kolmogorov-Smirnov test. Differences between two groups were compared using unpaired two-tailed t-tests or one-way analysis of variance, combined with Tukey's multiple comparison tests for post hoc comparisons. We performed power analyses using G*Power version 3 software (Universität Düsseldorf, Düsseldorf, Germany) 59 with one-way analysis of variance, and the two-sided significance level was set at α = 0.05. The effect size was evaluated in accordance with previous studies of the mouse bladder pain syndrome model. 60 Using nine animals, the statistical power was calculated at β > 0.95. The statistical analyses were performed using SPSS version 20 (IBM Corp., Armonk, NY, USA) and GraphPad Prism version 5 (GraphPad Software, La Jolla, CA, USA). Analysis items with two-tailed p values <0.05 were considered significant.

| CONCLUSIONS
To the best of our knowledge, this is the first in vitro and in vivo study to investigate the therapeutic potential of CNPs in UCPPS. Our study showed that CNPs are well tolerated, without inducing toxicity in T24 cells and no serious adverse events were observed in vivo mice. Further, CNP usage in a multimodal analgesic strategy may provide analgesic effects without serious risk of side effects, warranting further exploration. The ROS scavenging and downregulation of CXCL10 in the bladder tissue may play an immunomodulatory role in the management of UCPPS, and the downregulation of SerpinB2 may be also associated with decreased CXCL10 expression in the CYP cystitis mouse model pretreated with CNP. Further work is needed to explore the duration of CNP pretreatment for protection against UCPPS development. Our work supports CNP administration as a potential preventative strategy for patients at higher risk for UCPPS.